The overall objective of this proposal is to further our understanding of how energy conversion is accomplished in cytochrome c oxidase (CcO) and how rate and efficiency are controlled. In this grant period the PI proposes to continue the analysis of the structural basis of proton and electron transfer, using our model system R. sphaeroides CcO (RsCcO). The PI will take advantage of the exciting breakthrough by So Iwata's group in crystallizing RsCcO and several mutants, through collaboration with the Uppsala group to crystallize key mutants she has prepared. To facilitate all the crystallography efforts on RsCcO, the PI proposes to modify the over-producing strain by cloning and adding in the gene for the previously undetected Rs COX IV, a subunit that appears to be involved in crystal contacts. Another model system will also be developed by expressing in R. sphaeroides the cyanobacterial aa3 oxidase, whose unique properties and eukaryotic-like COX IV will allow the applicant to study some of the control features observed in the mammalian enzyme. The specific aims are: 1) To test various models of proton pumping and respiratory control by defining where "outside" is located in CcO, using stopped-flowed freeze-quench Mn ESEEM to measure deuterium access rates, and identifying residues and conformational features that control both access and activity; 2) To pursue the crystallization of key mutants by cloning and adding in the COX IV gene to the applicant's multi-copy plasmid to provide a strain of R. sphaeroides for making oxidase and mutant forms with a high probability of crystallizing; 3) To determine the critical factors governing electron transfer in CcO by developing new ruthenated versions of CcO and Cc to better analyze intrinsic electron transfer kinetics, and utilizing new methods for measuring redox potentials of mutants of CuA ligands and CuA/heme a interface; 4) To express Synechocystis cytochrome c oxidase (SynCcO) in R. sphaeroides and analyze its properties to determine the importance of missing key residues (e.g., Glu286), the nature of its unusual Cc interaction domain, and the regulatory significance of its eukaryotic-like COX IV. The PI expects that knowledge gained by these studies will lead to a clearer understanding of the mechanism of coupling, uncoupling and the control of efficiency of energy transduction, with important implications for weight control in aging and obesity.